Dendritic cells (DCs) comprise a diverse family of cell types whose primary function is to initiate and drive immune responses. Myeloid dendritic cells patrol areas of the body that are susceptible to invasion by pathogens and engulf antigens for later processing and presentation to T lymphocytes. HIV has appropriated this feature of the immune system to better establish and maintain infection of its primary target--CD4 positive T cells. DCs can efficiently bind and transfer HIV infection without themselves becoming infected, a process called trans-infection, and can in fact greatly enhance the infection of CD4 T cells. DCs are therefore potentially important in initiating HIV infections and in the persistence of viral reservoirs in infected individuals, making them an attractive target for new therapies aimed at prevention and treatment of HIV infection. Using high resolution live-cell microscopy, my lab tracks the trafficking of green fluorescent protein (GFP) tagged HIV within and between DCs and T- cells during trans-infection. We have previously reported that DCs enhance HIV infection by concentrating the virus to sites of contact with CD4 T cells at the same time that the HIV receptors (CD4 and CXCR4/CCR5) are concentrated on the surface of the T cell, forming a structure we called the infectious synapse. More recently, we have discovered that HIV is routed through a unique, pocket- like intracellular structure within DCs prior to transmission to the T cell, and that individual particles are delivered from the pocket and efficiently fuse into the T cell at the infectious synapse. The experiments proposed here will define the molecular machinery involved in the formation of this pocket structure in DCs and how HIV escapes from the pocket at the infectious synapse. We will test the role of DC trans- infection in cultured lymphoid tissue explants using novel fluorescently tagged HIV strains that allow us to track HIV dissemination among infected cell populations. Using specific chemical and molecular inhibitors, we will identify key regulators of trans-infection and explore the relationship between antigen presentation and trans-infection with the goal of tipping the balance away from trans-infection and toward degradation and immune presentation of the virus. We will apply the knowledge gained in the second phase of this study to intervene in trans-infection in the tissue explant model, with the goal of developing therapeutic strategies that reduce DC mediated HIV dissemination and enhance immune control. PUBLIC HEALTH RELEVANCE: During the course of HIV disease, the human immune system engages in a pitched battle with the virus, however the potent CD4 T cell responses that are elicited are the very thing that provides the fuel for HIV infection. Dendritic cells initiate protective immunity by presenting viral antigens to CD4 T cells, however they also elicit destructive infection of T cells through trans-infection. Our studies will define the pathways that distinguish immune presentation and viral dissemination by DCs and provide a framework for understanding these interactions in living tissues. The long-term goal of this study is to discover new drug targets to intervene in the transmission of HIV by DCs, shifting the balance away from trans-infection in favor of immune presentation and control of HIV disease.